The present invention relates to a novel endo-xcex2-N-acetylglucosaminidase gene, and specifically relates to a gene where said gene is derived from the genus Mucor. Further, the present invention relates to a recombinant plasmid which comprises said gene, an organism transformed with said plasmid, and a method of producing a novel endo-xcex2-N-acetylglucosaminidase using said transformant.
Glycoproteins are widely found in animal tissue, plant tissue, and the cell membrane and cell wall etc. of eukaryotic microorganisms.
In recent years, it has become increasingly clear that the sugar chains of glycoproteins play an important role in mechanisms such as cell differentiation, carcinogenesis and intercellular recognition. In order to clarify these mechanisms, research into the correlation between sugar chain structure and its function is proceeding. As a means of achieving this objective, when cleaving a sugar chain from a glycoprotein or when identifying the structure of a sugar chain, various glycosidases are employed. Among these, endo-xcex2-N-acetylglucosaminidase acts on the asparagine-linked sugar chain (N-linked sugar chain, N-sugar chain) and has the action of cleaving the diacetyl-chitobiose portion that exists within the sugar chain thereby liberating the sugar chain.
Since endo-xcex2-N-acetylglucosaminidase can liberate the sugar portion of a glycoprotein from the protein portion, it is thought to be important in the analysis of the function and structure of sugar chains in glycoproteins.
Asparagine-linked sugar chains may be classified by their structure as high mannose type (mannane type sugar chain), hybrid type, or complex type.
Known endo-xcex2-N-acetylglucosaminidases include Endo H (A. L. Tarentino and F. Maley, J. Biol. Chem., 249, 811 (1974)), Endo F (K. Takegawa, et al., Eur. j. Biochem., 202, 175 (1991)), and EndoA (K. Takegawa, et al., Appl. Environ. Microbiol., 55, 3107 (1989)). However, these enzymes only act upon sugars with specific structures, and do not act upon glycoproteins except in the presence of a denaturing agent.
Endo-xcex2-N-acetylglucosaminidase derived from Mucor hiemalis is capable of cleaving tri-antennary complex type sugar chains in respect of not only high mannose type (mannane type sugar chain) and hybrid type, but also complex type chains. Further, with the asialylated type, cleavage ability extends to tetra-antennary heteroglycan chains. Further, it is known that it is possible to free sugar chains from glycoproteins without subjecting the protein to denaturization treatment. (S. Kadowaki, et al., Agric. Biol. Chem., 54, 97 (1990)). Therefore, endo-xcex2-N-acetylglucosaminidase derived from Mucor hiemalis can be said to be useful in the research of the functional and physiological role of sugar chains and proteins of glycoproteins.
On the other hand, conversion of mannane type sugar chains derived from yeast into sugar chains in a form compatible with humans, is extremely significant industrially. As methods for this conversion, in vivo conversion through improvement of the yeast sugar chain biosynthetic system by genetic manipulation, and in vitro conversion using the trans-glycosylation reaction can be considered. For the purpose of sugar conversion, endo-xcex2-N-acetylglucosaminidase is required to have as properties, 1) the substrate specificity, i.e an ability to cleave both mannane and complex types; and, 2) the ability to perform the trans-glycosylation reaction, which is the reverse reaction of the decomposition reaction. Therefore, endo-xcex2-N-acetylglucosaminidase derived from Mucor hiemalis can be said to be an appropriate enzyme for the practice of said conversion.
The present inventors, have proposed a sugar chain conversion technique using endo-xcex2-N-acetylglucosaminidase derived from Mucor hiemalis which can alter yeast sugar chains to human-compatible form. (Japanese Patent Application Laid-Open No: Hei 7-59587)
To perform sugar chain conversions such as the above, an authentic enzyme preparation of high purity is required in great amounts. In this case, improvement of enzyme productivity by conventional breeding methods using mold cells has been considered. However, since conventional breeding methods are limited predominantly to the method of selecting such an enzyme from mutant strains obtained using ultraviolet light or mutagens, the isolation of stable mutants is difficult. Further, conventional breeding methods are often accompanied by unfavorable transformations. Further, since molds generally produce protease enzymes, they are unfavorable for production of an enzyme for the purpose of sugar conversion. Since, in order to overcome these problems it is necessary to proceed through a number of purification steps, the work is complicated and the yield is low. For example, culturing a microorganism belonging to the genus Mucor which is a type of filamentous mold, even if purification of the supernatant of this culture is performed, contamination with protease cannot be prevented and preparation in large amounts is difficult because of low enzyme productivity of the microorganism, and thus this method was of little practical value.
Given the above, it is desired for the purpose of mass producing endo-xcex2-N-acetylglucosaminidase, that the gene for said enzyme be obtained and produced through the use of genetic engineering. Further, if the gene can be obtained, it can be expected that an enzyme with improved heat resistance and pH resistance and increased reaction rate can be obtained using protein engineering techniques. However, there have been no reported attempts at gene cloning to date.
It is an object of the present invention to provide an endo-xcex2-N-acetylglucosaminidase, an endo-xcex2-N-acetylglucosaminidase gene, a recombinant vector which comprises said gene, an organism transformed with said vector, and a method of producing endo-xcex2-N-acetylglucosaminidase.
The present inventors, as a result of their intensive research directed to solving the above problem, based on partial amino acid sequence information of said endo-xcex2-N-acetylglucosaminidase derived from Mucor hiemalis, have succeeded in obtaining the gene that encodes said enzyme from a cDNA library prepared from Mucor hiemalis which is a bacteria which produces said enzyme, have further succeeded in expressing this gene in yeast, and thereby completed the present invention.
Thus, the present invention provides the recombinant protein of (a) or (b) below:
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3; and,
(b) a protein comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 3 by deletion, substitution, insertion, or addition of at least one amino acid and having the activity of endo-xcex2-N-acetylglucosaminidase.
Further, the present invention provides a endo-xcex2-N-acetylglucosaminidase gene which encodes the protein of (a) or (b) below, and a gene that hybridizes with said gene under stringent conditions, and which comprises DNA encoding a protein that has endo-xcex2-N-acetylglucosaminidase activity.
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3; and,
(b) a protein comprising an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 3 by deletion, substitution, insertion, or addition of at least one amino acid and having the activity of endo-xcex2-N-acetylglucosaminidase.
Further, the present invention provides a gene comprising the DNA of (c) or (d) below:
(c) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2; and,
(d) a DNA which hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2, and encodes a protein having the activity of endo-xcex2-N-acetylglucosaminidase.
Said gene includes a gene derived from a microorganism belonging to the genus Mucor. 
Further, the present invention provides a recombinant vector which comprises said gene.
Further, the present invention provides a transformant which comprises said recombinant vector.
Further, the present invention provides a method for producing an endo-xcex2-N-acetylglucosaminidase, comprising culturing said transformant, and collecting endo-xcex2-N-acetylglucosaminidase from the obtained culture product.
Below, the present invention will be described in detail.
The present invention is characterized by culturing endo-xcex2-N-acetylglucosaminidase producing microorganisms, purifying the endo-xcex2-N-acetylglucosaminidase from the obtained culture, designing degenerate probes from a partial amino acid sequence of said enzyme, cloning a gene encoding said enzyme by performing PCR, and further, cloning a gene encoding said enzyme from a cDNA library of the microorganism producing endo-xcex2-N-acetylglucosaminidase. Further, the present invention is characterized by obtaining a recombinant vector by introducing the cloned gene into a vector, as well as by obtaining a transformant by introducing said recombinant vector into a host cell. Further, the present invention is characterized by producing endo-xcex2-N-acetylglucosaminidase in large quantities by culturing said transformant.
Microorganisms for producing endo-xcex2-N-acetylglucosaminidase include microorganisms belonging to the genus Mucor, preferably Mucor hiemalis, and more preferably the Mucor hiemalis deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japan) (Accession No. FERM BP-4991).
The medium composition used in culturing of these strains may be of any kind typically used in the culture of microorganisms.
Carbon sources include for example sugars such as glucose, sucrose, mannose, galactose, maltose, soluble starch and dextrin. Nitrogen sources include yeast extract, trypton, etc. As inorganic salts, apart from the inorganic salts contained in the above nitrogen sources, salts such as all varieties of sodium salts, potassium salts, calcium salts, magnesium salts and phosphate salts may be used. Vitamins may be added optionally.
The culture medium is sterilized by a conventional method, and the strain is inoculated into the medium. Thereafter, a shake culture, or aeration-agitation culture is performed at 20-30xc2x0 C., pH 5-7 for 2 to 4 days.
In the present invention, culture is preferably performed at 25-30xc2x0 C., pH 6, with galactose as the carbon source, yeast extract and trypton as the nitrogen sources, with concentrations of both carbon and nitrogen sources at 2-3% each, and the ratio of carbon source to nitrogen source at 2:3, for 3 to 4 days under good aeration conditions. Where the culture is performed under such conditions, the amount of enzyme produced is maximized, and in comparison to the known method (S. Kadowaki, et al., Agric. Biol. Chem., 54, 97 (1990); glucose 0.5%, yeast extract 1%, peptone 1%), about 10-fold greater productivity can be achieved.
Further, in the present invention, to ensure the aeration conditions when culturing the microorganisms, use of a jar fermenter is preferable.
The endo-xcex2-N-acetylglucosaminidase produced by the above bacterial strain is characterized by preservation of the following activity. In other words, it can be characterized by its activity to act on the asparagine-linked sugar chain in a glycoprotein, cleave the diacetyl-chitobiose portion within the sugar chain, and thereby liberate the sugar chain.
Purification of endo-xcex2-N-acetylglucosaminidase can be performed by the appropriate combination of known methods for separation and purification. Examples of such methods include methods using differences in solubility such as salt precipitation and solvent precipitation; methods using differences in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide electrophoresis; methods using differences in electrostatic charge such as ion exchange chromatography; methods using differences in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography, and methods using differences in isoelectric point such as isoelectric focusing.
In the present invention, by adopting a method of culturing which is an improvement over the above described known method (S. Kadowaki, et al., Agric Biol. Chem., 54, 97 (1990)), and subjecting to many purification steps, endo-xcex2-N-acetylglucosaminidase can be efficiently purified, and it is possible to obtain a sufficient amount of the protein to determine the amino acid sequence necessary to obtain the gene. The obtained enzyme, as a result of purification and as a result of the analysis of the gene to be described below, consists of a single gene product with a molecular weight of approximately 85,000, and after post-translational partial digestion of the gene product, it was found to be composed of 2 or more subunits including peptides with molecular weights of approximately 60,000 and 14,000.
It was clear that the endo-xcex2-N-acetylglucosaminidase obtained from Mucor hiemalis was composed of at least 2 or more peptides.
Generally, when isolating a gene encoding a specific protein, a partial amino acid sequence of the protein is determined, and it is possible to isolate the desired gene from a gene library with a mixture of oligonucleotides consisting of degenerative codons as a probe. Further, after obtaining a fragment by PCR such as in the present invention, it is possible to isolate the desired gene from a gene library using this fragment as a probe.
However, since endo-xcex2-N-acetylglucosaminidase is a hetero-oligomeric molecule consisting of 2 or more subunits, there is the possibility that each subunit is encoded independently by their separate differing genes. Further, even if endo-xcex2-N-acetylglucosaminidase is derived from one gene, its structure, for example, the positional relationship between the regions encoding the two subunits within the structural gene was unclear.
Thus, the present inventors determined partial amino acid sequences for 2 subunits, and then after obtaining partial fragments by PCR, used said fragments as probes to achieve cDNA cloning and by analyzing the gene structure, clarified that the same gene coded for these 2 subunits. That is, it was clarified that the novel endo-xcex2-N-acetylglucosaminidase is produced as one polypeptide from the gene encoding this enzyme, and is processed by partial decomposition into 2 or more subunits.
The gene of the present invention is cloned by, for example, the following method.
(1) Cloning of the Endo-xcex2-N-acetylglucosaminidase Gene
In the present invention, an example of a DNA fragment comprising a gene encoding a novel endo-xcex2-N-acetylglucosaminidase is the DNA fragment indicated by the restriction enzyme map shown in FIG. 2. This fragment can be isolated using genetic engineering methods from a cDNA library with an mRNA template prepared from a microorganism belonging to the genus Mucor, preferably from a strain of Mucor hiemalis, and more preferably from the strain of Mucor hiemalis deposited under accession number FERM BP-4991 at the National Institute of Bioscience and Human-Technology, (See the method described in, for example, Molecular Cloning: A Laboratory Manual (Sambrook, Maniatis et al, Cold Spring Harbour Laboratory Press (1989))
Preparation of mRNA can be performed according to a typical method. For example, after culturing the mRNA source, Mucor hiemalis, total RNA is obtained from the cultured cell with a kit available on the market (ISOGEN (Nippon Gene Company)), and then purified with a purification kit available on the market (mRNA Purification Kit (Pharmacia Biotech)). In the preparation of mRNA, it is preferable to keep the culturing time short to control the decomposition of mRNA.
With the thus obtained mRNA as a template, a single-strand cDNA is synthesized using an oligo dT primer and a reverse transcriptase enzyme. Thereupon, a duplex cDNA is synthesized from said single stranded cDNA. A recombinant vector is then constructed by incorporating the duplex cDNA into a suitable cloning vector. A cDNA library can be obtained by transforming E.coli using the obtained recombinant vector, and selecting or transformants using tetracycline resistance and ampicillin resistance as an indicator.
Here, transformation of E.coli is performed according to a method such as the Hanahan method [Hanahan, D.: J. Mol. Biol. 166: 557-580 (1983)]. When a plasmid is to be used as a vector, it is necessary to include a gene for resistance to a drug such as tetracycline or ampicillin. Further, a cloning vector other than a plasmid, for example, xcex phage or the like can be used.
A strain having the desired DNA is selected (screened) from the thus obtained transformant. Screening methods include, for example, a method of synthesizing sense and antisense primers corresponding to a partial amino acid sequence of endo-xcex2-N-acetylglucosaminidase, and using this to perform a polymerase chain reaction (PCR). Template DNA may include for example, genomic DNA or cDNA synthesized by reverse transcription from the above-mentioned mRNA. As primers, in respect of the sense chain, for example, 5xe2x80x2-CARTTRCARCCNGAYGAYAA-3xe2x80x2 (SEQ ID NO: 5) synthesized on the basis of amino acid sequence: PSLQLQPDDK (SEQ ID NO: 4) and 5xe2x80x2-CCHACNGAYCARAAYATYAA-3xe2x80x2 (SEQ ID NO: 7) synthesized on the basis of amino acid sequence: SYRNPEIYPTDQNIK (SEQ ID NO: 6), may be used. Further, in respect of the antisense chain 3xe2x80x2-GGDTGNCTRGTYTTRTARTT-5xe2x80x2 (SEQ ID NO: 8) synthesized on the basis of amino acid sequence: SYRNPEIYPTDQNIK (SEQ ID NO: 6) and 3xe2x80x2-TTYCCDGTYGCDAARTTRGT-5xe2x80x2 (SEQ ID NO: 10) synthesized on the basis of amino acid sequence: GQRFNHRESHDVETEI (SEQ ID NO: 9), may be used. However, the present invention is not limited to these primers.
Thus, the obtained DNA amplification fragment is labeled with for example 32P, 35S or biotin and taken as a probe, and is then made to hybridize with the cDNA library of the transformant, which library has been denatured and immobilized onto a nitrocellulose filter. Screening can then be performed by searching the obtained positive strains.
(2) Determination of the Nucleotide Sequence
Determination of the nucleotide sequence of the obtained clone is performed. Determination of the nucleotide sequence may be performed by known methods such as the Maxam-Gilbert chemical modification method or dideoxy method, though typically determination of the sequence is performed using an automated nucleotide sequencer. (For example, PERKIN-ELMER 377A DNA Sequencer)
The full length sequence of the endo-xcex2-N-acetylglucosaminidase gene is indicated by SEQ ID NO: 1. Therein, a preferable example of the gene of the present invention, is the nucleotide sequence from position 71 to position 2305 (SEQ ID NO: 2) of the nucleotide sequence indicated by SEQ ID NO: 1. Further, the gene of the present invention includes not only a sequence which encodes the amino acid sequence represented by SEQ ID NO: 3 or the below described amino acid sequence having an equivalent sequence, but also encompasses degenerate isomers encoding identical polypeptides which differ in respect of degenerate codons only.
A nucleotide sequence encoding an amino acid sequence having an equivalent sequence can be prepared using a method such as site-directed mutagenesis. That is, mutations may be introduced by known methods such as the Kunkel method or Gapped duplex method, or other methods equivalent thereto, using for example a mutation introduction kit that employs site-directed mutagenesis (e.g. Mutant-K (Takara), Mutant-G (Takara)) etc., or using Takara""s LA PCR in vitro Mutagenesis series kit.
Further, an endo-xcex2-N-acetylglucosaminidase gene includes not only DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, but also a DNA which hybridizes under stringent conditions with said DNA, and encodes a protein having the activity of endo-xcex2-N-acetylglucosaminidase. Stringent conditions refer to, for example, the conditions of a sodium concentration of 50-300 mM, preferably 105 mM and a temperature of (50-68xc2x0 C., preferably 65xc2x0 C.
Once the nucleotide sequence of the endo-xcex2-N-acetylglucosaminidase gene (SEQ ID NO: 1) is identified, since the nucleotide sequence of the DNA fragment having the sequence from position 71 to 2305 of the said nucleotide sequence (open reading frame) is determined (SEQ ID NO: 2), it is possible to obtain the endo-w -N-acetylglucosaminidase gene by chemical synthesis; by PCR with genomic DNA as a template and the 5xe2x80x2 and 3xe2x80x2 terminal sequences of the open reading frame (SEQ ID NO: 2) (e.g. 5xe2x80x2-ATGCCTTCACTTCAATTGCAACC-3xe2x80x2 (SEQ ID NO: 11) and 5xe2x80x2-CTAGTTTAATGACAAATCTATGC-3xe2x80x2 (SEQ ID NO: 12) as primers; or, by hybridization with a DNA fragment having the nucleotide sequence of an endo-xcex2-N-acetylglucosaminidase gene as a probe.
The plasmid pZL-Endo (See Example 3 below) which comprises the gene of the present invention was introduced into E.coli DH10B (Title: DHBpZL-Endo) and deposited at National Institute of Bioscience and Human-Technology (1-1-3, Higashi, Tsukuba-shi, Tbaraki-ken, Japan) on Apr. 28, 1998 under accession number FERM BP-6335.
In the present invention, the amino acid sequence represented by SEQ ID NO: 3 or a polypeptide comprising an equivalent sequence, is provided as a preferable example of a recombinant novel endo-xcex2-N-acetylglucosaminidase. Here, xe2x80x9cequivalent sequencexe2x80x9d refers to a sequence which comprises the amino acid sequence represented by SEQ ID NO: 3 and in which at least one amino acid is inserted, substituted, deleted or added to either end, and which retains said novel endo-xcex2-N-acetylglucosaminidase activity. Retention of novel endo-xcex2-N-acetylglucosaminidase activity in this equivalent sequence means that the sequence maintains activity sufficient for it to be used in an almost identical manner under identical conditions to the polypeptide having the full sequence represented by SEQ ID NO: 3 in actual forms of use which exploit this activity. It is clear that it is possible for a person skilled in the art to select and produce with no particular difficulty such an equivalent sequence; referring to the sequence represented by SEQ ID NO: 3. For example, within the amino acid sequence represented by SEQ ID NO: 3, at least 1, preferably 1 to 10, more preferably 1 to 5 amino acids may be deleted; at least 1, preferably 1 to 10, more preferably 1 to 5 amino acids may be added or inserted; or, at least 1, preferably 1 to 10, more preferably 1 to 5 amino acids may be substituted. Accordingly, the protein of the present invention includes a polypeptide having the amino acid sequence from position 2 to 744 of the amino acid sequence represented by SEQ ID NO: 3 in the Sequence Listing (one in which the methoinine at position 1 of the amino acid sequence represented by SEQ ID NO: 3 has been deleted.) Here, through the partial amino acid sequence analysis of the present invention and gene structure analysis, it became clear that 2 or more natural type subunits were produced by cleavage of a precursor polypeptide on the C terminal side of at least the histidine at position 510 and the asparagine at position 627 in the amino acid sequence represented by SEQ ID NO: 3.
The present invention provides a DNA molecule comprising the gene of the present invention, in particular, an expression vector. This DNA molecule can be obtained by incorporating a DNA fragment encoding the novel endo-xcex2-N-acetylglucosaminidase according to the present invention into a vector molecule. Accordingly, if transformation of a host cell is performed with a DNA molecule, particularly in the form of an expression vector, which includes a DNA fragment encoding the novel endo-xcex2-N-acetylglucosaminidase of the present invention in a form such that it is replicable within the host cell and the said gene is expressible, it is possible to cause production of the novel endo-xcex2-N-acetylglucosaminidase of the present invention in the host cell.
The DNA molecule according to this invention can be constructed based on the method described in the above-referenced Molecular Cloning: A Laboratory Manual (supra).
(1) Construction of a Recombinant Vector
The vector to be used in the present invention may be appropriately selected from a virus, plasmid, cosmid vector or the like in consideration of the type of host cell to be used.
For example, where the host cell is E.coli, bacteriophages of the xcex phage line, plasmids of the pBR line (pBR322, pBR325 etc.) and pUC line (pUC118, pUC119 etc); where the host cell is Bacillus subtilis, plasmids of the pUB line (pUB110 etc); and where the host cell is yeast, vectors of the YEp and YCp lines (e.g. YEp13, YEp24, YCp50 etc.), or pG-3-Not used in the Examples below, may be used. Further, animal viruses such as a retrovirus or vaccinia virus, or insect virus vectors such baculovirus can be used.
To introduce the gene of the present invention into the vector, methods for ligating the gene to the vector such as first cleaving the purified DNA with a suitable restriction enzyme, and then inserting the gene at suitable restriction sites or multicloning sites of the vector DNA, are employed.
It is necessary that the gene of the present invention be incorporated into the vector such that the function of this gene is exhibited. Thus, it is preferable that the vector of the present invention includes a selective marker. Drug-resistant markers and auxotrophic markers can be used as selective markers.
Further, it is preferable that the DNA molecule to be used as the expression vector of the present invention has DNA sequences necessary for the expression of the novel endo-xcex2-N-acetylglucosaminidase gene such as transcription regulating signals and translation regulating signals such as, for example, a promoter, transcription initiation signal, ribosome binding site, translation stop signal and a transcription termination signal.
(2) Construction of the Transformant
The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host in a manner allowing expression of the subject gene. There is no particular limitation on the host that may be used as long as it allows expression of the gene of the present invention. Examples include bacteria of the genus Escherichia such as Escherichia coli, of the genus Bacillus such as Bacillus subtilis, or of the genus Pseudomonas such as Pseodomonas putida etc., and yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida boidinii and Pichia pastoris. 
As host cells, apart from E.coli, B. subtilis, and yeast, animal cells such as COS cells and CHO cells etc., and insect cells such as Sf9 and Sf21 etc. may be used.
Where a bacterium such as E.coli is used as a host, the recombinant vector of the present invention preferably is autonomously replicable within the bacterium and comprises a promoter, ribosome binding site, the gene of the present invention, and a transcriptional termination sequence. A gene which controls the promoter may also be included.
Examples of E. coli include Escherichia coli K12, DH1, DH5xcex1, JM109 etc. Examples of Bacillus subtilis include Bacillus subtilis MI 114, 207-21 etc. It is known that there exist strains of Bacillus subtilis that secrete proteins out of the microorganism body. There are also known strains that secrete hardly any protease. The use of such strains as hosts is preferable.
As a promoter, a promoter within the inserted fragment that is able to function even in the host, may of course be used. Examples of promoters in E.coli include lactose operon (Oac) and tryptophan operon (trp), etc.
As a method for introducing the recombinant vector into bacteria, any method for introducing DNA into bacteria may be employed and there is no particular limitation thereon. Examples include a method using calcium ions [Cohen, S.N. et al.: Proc. Natl. Acad. Sci., USA, 69:2110-2114(1972)], and electroporation.
Where yeast is to be the host, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, etc., may be used. In this case, there is no particular limitation on the promoter that may be used as long as it can express in yeast. For example, promoters such as alcohol dehydrogenase (ADH), acidic phosphatase (PHO), galactose gene (GAL),and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) promoters, heat shock protein promoter, MFxcex11 promoter, PGK promoter, GAP promoter and AOX1 promoter may preferably be used.
As a method for introducing the recombinant vector into yeast, any method for introducing DNA into yeast may be employed and there is no particular limitation thereon. Examples include electroporation [Becker, D. M. et al.: Methods. Enzymol., 194: 182-187 (1990)], spheroplast method [Hinnen, A. et al.: Proc. Natl. Acad. Sci., USA, 75: 1929-1933 (1978)], and lithium acetate method [Itoh, H.: J. Bacteriol., 153:163-168 (1983)].
Where an animal cell is to be the host, monkey cell COS-7, Vero, Chinese hamster ovary cell (CHO cell), mouse L cell, rat GH3 and human FL cell, etc. may be used. As a promoter, SR xcex1 promoter, SV40 promoter, LTR promoter, CMV promoter or the like can be used. Also, the initial promoter of human cytomegalovirus or the like may be used.
Examples of a method for introducing the recombinant vector into an animal cell include electroporation, calcium phosphate method and lipofection method.
Where an insect cell is to be the host, Sf9 cell, Sf21 cell and the like may be used.
Examples of a method for introducing the recombinant vector into an insect cell include calcium phosphate method, lipofection, and electroporation.
A protein of the present invention has an amino acid sequence encoded by a gene of the present invention, or has said amino acid sequence into which said modification of at least 1 amino acid has been introduced, and has the activity of endo-xcex2-N-acetylglucosaminidase.
The protein of the present invention can be obtained by culturing the above-mentioned transformant and collecting the protein from this culture product. xe2x80x9cCulture productxe2x80x9d refers to either the culture supernatant, the cultured cells or microbial cells, or disrupted cells or microbial cells.
Either a natural medium or synthetic medium may be used as a medium for culturing transformants obtained with microorganisms such as E. coli or yeast as hosts, as long as it contains a carbon source, a nitrogen source, inorganic salts and the like, which are able to be assimilated by the microorganism and it may be used to efficiently perform a culture of the transformant.
As carbon sources, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.
As nitrogen sources, inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, organic acids such as ammonium acetate, or other nitrogen containing compounds, as well as peptone, meat extract, corn steep liquor or the like are used.
As inorganic matter, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron (I) sulfate, manganese sulfate, copper sulfate and calcium carbonate, etc are used.
Culturing is typically performed under aerobic conditions such as a shake culture or aeration-agitation culture, at 37xc2x0 C. at 12 to 72 hours. During culturing, pH is maintained at 4-7.5. Regulation of pH is performed using inorganic or organic acid or alkali solutions and the like.
During culturing, antibiotics such as ampicillin and tetracycline may be added to the medium as required.
When the microorganism transformed with an expression vector containing an inducible promoter is cultured, an inducer may he added to) the culture as required. For example, when culturing a microorganism transformed with an expression vector which uses a Lac promoter, isopropyl-xcex2-D-thiogalactoside (EPIG) and the like may be added to the culture, and when culturing a microorganism transformed with an expression vector using a trp promoter, indole acetic acid (IAA) may be added.
As culture media for culturing a transformant obtained from an animal host cell, the generally used RPMI1640 medium, DMEM medium or these medium to which fetal calf serum and the like have been added, are used.
Culturing is typically performed over 2 to 10 days at 37xc2x0 C. in the presence of 5% CO2. Antibiotics such as kanamycin and penicillin and the like may be added during culturing as required.
After culturing, when the protein of the present invention has been produced within the microorganisms or cells, the protein of the present invention is extracted by disrupting the microorganisms or cells. When the protein of the present invention has been produced outside the microorganisms or cells, the culture fluid may be used as is, or the microorganisms or cells removed by centrifugal isolation, etc.
Purification of the recombinant novel endo-xcex2-N-acetylglucosaminidase, is performed by the suitable combination of known separation and purification methods. Examples of such methods include methods using differences in solubility such as salt precipitation, and solvent precipitation methods; methods using differences in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacryl electrophoresis; methods using differences in electrostatic charge (valence) such as ion exchange chromatography; methods using differences in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography; and, methods using differences in isoelectric point such as isoelectric focusing.
In the present invention, as indicated in the Examples below, when this gene was made to express under the control of GAPDH promoter, in a Saccharomyces cerevisiae host, high enzyme activity was confirmed within the cell extract. This indicated that it was possible to produce active novel endo-xcex2-N-acetylglucosaminidase in large quantities through the expression of the gene of the present invention in the recombinant.